Integrin binding motif containing peptides and methods of treating skeletal diseases

ABSTRACT

Peptide sequences comprising 10 to 50 amino acids are disclosed. The sequences are characterized by containing at least one of an integrin binding motif such as an RGD sequence, a glycosaminoglycan binding motif, and a calcium binding motif, and the remainder of amino acids contiguous with the RGD sequence in matrix extracellular phosphoglycoprotein. The sequences may be formulated for injection or dispersed in toothpaste or a mouthwash or gum patch and administered to enhance bone/tooth growth and/or reduce excessive urinary phosphate loss from the body.

CROSS-REFERENCE

This application is a continuation of application Ser. No. 11/531,583filed Sep. 13, 2006 which is a continuation of application Ser. No.11/042,492 filed Jan. 24, 2005 (now U.S. Pat. No. 7,160,862 issued Jan.9, 2007) which is a continuation of application Ser. No. 09/812,485filed Mar. 19, 2001 (now U.S. Pat. No. 6,911,425 issued Jun. 28, 2005)which is a continuation-in-part application of Ser. No. 09/641,034,filed Aug. 16, 2000 (now abandoned), which are incorporated herein byreference in their entirety and to which applications we claim priorityunder 35 USC § 120.

FIELD OF THE INVENTION

The invention relates generally to the field of peptides and moreparticularly to peptides and formulations thereof useful in treatingskeletal diseases.

BACKGROUND OF THE INVENTION

It is well-documented that disorders of skeletal tissues and mineralmetabolism cause numerous significant health problems on world-widebasis.

In humans, the maximum bone mass occurs between the age of 15 and 40 andis referred to as “peak bone mass.” After such peak bone mass age, bonemass begins declining gradually and the mechanical strength of the boneis accordingly reduced. Consequently, when mechanical strength declinesto a certain level, the individual is at greater risk of bone fracture.This natural occurrence is called osteoporosis if severe enough to bepathogenic.

The speed at which bone loss occurs differs among individuals, andespecially with respect to gender. In females, the speed of bone lossaccelerates immediately after menopause (See FIG. 1) because of asignificant decline in available estrogen, a hormone which plays acritical role in maintaining healthy bone metabolism. Postmenopausalosteoporosis constitutes an important clinical problem because itafflicts significant numbers of women. Notably, the ratio of female tomale osteoporosis patients is 3:1.

The majority of bone diseases are characterized by loss of boneminerals, weakening of bones and consequently, an increase of thefrequency and severity of bone fractures, which are called “pathologicalfracture.” In the elderly population, this has significant socialramifications as well, as many of those with bone fractures havedifficulty with mobility, which often leads to the deterioration ofother mental and physical functions, resulting in dementia, muscularweakness and/or fatigue. In addition, morbidity and pain aresignificantly increased by thrombotic events, such as pulmonary embolismwhich can occur as a result of hip or pelvic fractures.

In the United States alone, it is said that 52 million women over age of45 will suffer from osteoporosis by 2000. Current worldwide osteoporosispopulation is around 200 million. Annual incidence of pathologicalfracture in the United States alone is approximately 1.5 million. It isestimated that annual medical costs for those osteoporosis patients inthe United States and world are $14 billion and $60 billion,respectively.

Renal failure is also a significant health problem related to mineralmetabolism and skeletal formation, and the number of its patients isincreasing rapidly. Renal function is declining gradually over severalto ten years period in these patients. When the renal function becomesapproximately a quarter (¼) of the healthy level, the patients areclassified to chronic renal failure. When it becomes approximately onesixth (⅙) thereof, they need to start dialysis and are called end stagerenal disease (ESRD). In patients with chronic renal failure, serumlevels of important minerals such as calcium and phosphate lose theirnormal homeostasis, which results in malformation of skeleton. It iscalled renal osteodystrophy (ROD), which is a secondary osteoporosisfrom renal failure. ROD can also cause pathological fracture likeosteoporosis. The prevalence of end stage renal disease (ESRD) in theUnited States is rapidly increasing and about to reach 300 thousand in2000. ROD affects most ESRD patients.

There are several other diseases of skeletal tissues and mineralmetabolism such as Paget's Disease, rickets, osteopetrosis,hyperparathyroidism, and so forth and a number of patients are affectedby these diseases.

Metabolically, bone is a highly active organ with bone resorption andformation occurring continuously (remodeling). Bone resorption isfacilitated by osteoclasts which are differentiated frommonocyte/macrophage lineage cells. Osteoclasts adhere to the surface ofbone and degrade bone tissue by secreting acids and enzymes. Osteoblastsfacilitate bone formation by adhering to degraded bone tissue andsecreting bone matrix proteins, which are mineralized mostly by calciumand phosphate. Osteoblasts differentiate into bone cells (osteocytes),and become a part of bone tissue.

Numerous experimental approaches have been attempted to eitheraccelerate bone formation or diminish bone resorption. For example,growth factors such as BMPs (bone morphogenetic proteins), TGFβ(transforming growth factor β), IGF (insulin-like growth factor), andfibroblast growth factor (FGF) are known to have potent biologicalactivities in bone formation. In particular, a few subfamily moleculesof BMP such as BMP-2 is regarded as one of the most potent growthfactors for hard tissue. However, these factors have not been developedas therapeutic agents for systemic bone diseases. It is because none ofthem can be delivered to the bone selectively and some of these factorssuch as BMPs convert soft tissue into hard tissue. It is called ectopiccalcification and is a critical adverse effect for them when they areused systemically. Further, the processes of bone formation andresorption are so closely connected and that makes selective increase ofbone formation or selective inhibition of bone resorption extremelydifficult.

Currently, there is a need for an effective treatment for bone loss.Therapeutic agents such as estrogen, calcitonin, vitamin D, fluoride,Ipriflavon, bisphosphonates, and a few others have failed to provide asatisfactory means of treatment. (Gennari et al., Drug Saf. (1994)11(3):179-95).

Estrogen and its analogues are frequently administered to patients withpostmenopausal osteoporosis. Estrogen replacement therapy involvesadministration of estrogen just prior to or after the onset ofmenopause. However, as is often the case with steroid hormones, the longterm use of estrogen has significant adverse effects such as breast andother gynecological cancers (Schneider et al., Int. J. Fertil.Menopausal Study (1995) 40(1):40-53).

Calcitonin, an endogenous hormone produced by the thyroid, bindsselectively to osteoclasts, via its receptor, and inactivates them.Since the osteoclast is the only cell which can dissolve bone tissue,calcitonin binding can block or slow down bone degradation caused by theosteoclast. However, this biological mechanism is very short-lived, asthe osteoclasts become tolerant to this drug relatively quickly.Therefore, the use of calcitonin does not provide an effectivetherapeutic option.

Fluoride has been shown to increase bone mass when it is administered tohumans. However, while bone mass is increased, mechanical strength isnot. Therefore, despite the increase in apparent bone mass, the risk offracture remains (Fratzl et al., J. Bone Mineral Res. (1994)9(10):1541-1549). In addition, fluoride administration has significanthealth risks.

Ipriflavon has been used to treat osteoporosis in limited areas in theworld. However, the actual efficacy of this compound is questionable andit is not widely accepted as a useful therapeutic agent for bonediseases.

Bisphosphonates are compounds derivatized from pyrophosphate. Synthesisinvolves replacing an oxygen atom situated between two phosphorus atomswith carbon and modifying the carbon with various substituents. Whilebisphosphonates are known to suppress bone resorption, they have littleeffect on bone formation. Furthermore, bisphosphonates adhere to thebone surface and remain there for very long time causing a long-termdecrease in bone tissue turnover. As bone tissue needs to be turned overcontinuously, this decrease in turnover ultimately results in bonedeterioration (Lufkin et al., Osteoporos. Int. (1994) 4(6):320-322;Chapparel et al., J. Bone Miner. Res. (1995) 10(1):112-118).

Another significant problem with the agents described above is that withthe exception of fluoride and ipriflavon, they are unsuitable for oraladministration, and thus, must be given parenterally. Since bonedisorders are often chronic and require long-term therapy, it isdesirable that therapeutic agents be suitable for oral administration.

In summary, a significant need exists for a therapeutic agent which canprevent or treat bone loss. In particular, a new drug that canselectively increase bone formation and/or number of osteoblast withoutaffecting bone resorption or soft tissue is highly desired.

Another major health problem relating to skeleton and mineral metabolismis that with teeth. In the United States alone, it is estimated that 67million people are affected by periodontal disease and that the annualcost of its treatment is approximately $6.0 billion in 2000. It is said90% of the entire population experience dental caries in their lives.The annual cost to treat them is over $50 billion per year in the UnitedStates alone.

Dental caries are a universal disease and affects children and adults.Periodontal disease, on the other hand, affects mostly adults, and inparticular, the aged. In many cases, the patient's gum is inflamed anddestroyed, and the alveolar bone that supports the teeth isdeteriorated. Cement that composes the core of the root is also damaged,and subsequently, teeth fall out. One of the most common treatments fortooth loss involves the use of a dental implant. An artificial implant(osseointegrated dental implants) is placed in the space where the toothwas lost. In severe cases, an entire denture is replaced by implants.However, implants frequently loosen, or fall out because their fixationon the alveolar bone is not always successful. Since alveolar bone issomehow damaged in these patients, the implant cannot always besupported well by alveolar bone. When alveolar bone is severely damaged,autogenous bone grafting is performed. In this case, a bone graft takenfrom another skeletal tissue of the same patient is grafted in thedamaged alveolar area so that the hard tissue is regenerated and sinusis elevated there. Since these treatments require expensivebio-compatible materials and/or highly skilled techniques, the cost oftreatment is usually very high.

It is believed that dental caries are caused by acidic condition in theoral cavity. For instance, sugars are converted to acid and dissolve thesurface of the teeth. Although only enamel and a part of dentin isaffected in many cases, the damage can reach the pulp cavity in severecases that cause significant pain. The most typical treatment is fillingthe caries lesion with non-degradable materials such as metals or metaloxide. Treatment of dental caries mostly depends upon those materialsand the techniques by the dentists, which is often expensive.

Although a few therapeutic agents have been developed and used in dentalarea, they are generally only anti-inflammatory drugs, analgesics, andantibiotics. No generally effective therapeutic agent that directlyimproves periodontal hard tissues has been developed.

Another major clinical problem in mineral metabolism is excessive lossor waste of phosphate (PO₄) out of the body system. Phosphate playsvariety of important roles in all living creatures. In vertebrates,phosphate is a major component of their skeleton. In all animals,phosphate is an essential component to build polynucleotide chains andcell membranes; phosphorylation and dephosphorylation of sugars andnucleotides are the most essential reactions in energy generation andconsumption; and phosphorylation and dephosphorylation of proteins,sugars, and lipids are indispensable reactions for signal transductionin the cells. Therefore, a shortage of phosphate could even result indeath.

In mammals, phosphate concentration in the body fluid is controlledwithin a range that allows all normal biological functions in the body.The kidney is the most important organ for controlling phosphate levelsin the body. Glomeruli in the kidney filter phosphate constantly to theurine, and proximal tubules usually reabsorb approximately 80% of thisfiltered phosphate. If this reabsorbing function is damaged, excessivephosphate is lost into the urine, resulting in various clinicalproblems.

For instance, it is well known that the majority of kidney transplantpatients experience excessive renal phosphate leakage, because thetransplanted kidneys only marginally reabsorb the urinary phosphate tothe circulation. The reasons for this poor reabsorbing activity on thepart of transplanted kidneys are unknown. It frequently causes thepatients malnutrition and secondary osteoporosis. This problem cannot betreated by a simple exogenous supplementation of phosphate. Similarrenal phosphate leakage with unknown pathology is often observed inpediatric medicine, with outcomes such as malnutrition or growthretardation.

Health problems associated with circulating phosphate shortage is notlimited to humans. Milking cows sometimes suffer from hypophosphatemia(too low phosphate in the blood) by overproduction of the milk. It notonly deteriorates the nutritional quality of the milk but also oftenmake the cows useless for milk production. It is as relatively commonproblem in dairy farms.

Clearly, there is a significant demand for a therapeutic agent thatpromotes regeneration of alveolar bone and/or teeth, increases thenumber and activity of odontoblasts/osteoblasts that help form dentaltissues, and reduces renal phosphate secretion.

SUMMARY OF THE INVENTION

A class of compounds is disclosed which are useful in treating orpreventing a condition associated with skeletal loss or weakness and/orwhich reduce renal phosphate excretion. The compounds are peptides oranalogs thereof which comprise between 10 and 50 monomer (e.g. aminoacids) units. The amino acid sequence comprises one or more of thefollowing motifs: an integrin binding motif sequence; aglycosaminoglycan binding motif; and a calcium-binding motif. The aminoacids may be in the D- or L-conformation. The remaining monomer units(the sequence other than the aforementioned motifs) in the compound maybe amino acid analogs. Where the motif is an integrin binding motif, theremaining monomer units are preferably naturally occurring amino acidshaving a sequence which are substantially the same as an amino acidsequence contiguous with the RGD sequence in the naturally occurringprotein, matrix extracellular phosphoglycoprotein (Rowe et. al.,Genomics (2000) 67:56-68).

An aspect of the invention is a set of peptides and/or peptide analogs.

A feature of the invention is that a compound of the invention comprisesone or more of the following motifs: an integrin binding motif sequence;a glycosaminoglycan-binding motif; and a calcium-binding motif. Theamino acids may be in the D- or L-conformation.

An advantage of the invention is that a compound of the inventionenhances skeletal growth.

Another advantage of the invention is that a compound of the inventionenhances the number of osteoblast and possibly odontoblast cells on thesurface of new skeletal or tooth growth.

Another advantage of the invention is that a compound of the inventionreduces phosphate (Pi) loss from the body, as indicated by reducedurinary Pi leakage.

Another aspect of the invention is to provide a formulation fortherapeutic use which comprises a sufficient concentration of a compoundof the invention and can be administered to the pulp of teeth, the spacebetween the root of teeth and gum, or alveolar bone to prevent thedamage on teeth and/or alveolar bone or regenerate the hard tissue inthe damaged teeth and/or alveolar bone.

Another aspect of the invention is to provide toothpaste which comprisesa sufficient concentration of a compound of the invention to enhancetooth and/or alveolar bone growth on areas where deterioration hasoccurred, or to prevent such deterioration.

Yet another aspect of the invention is to provide a mouthwash whichcomprises a sufficient concentration of a compound of the invention toenhance tooth and/or alveolar bone growth on areas where deteriorationhas occurred, or to prevent such deterioration.

Still another aspect of the invention is a dental floss having coatedthereon and/or embedded therein a compound of the invention in an amountsuch that repeated application to teeth and/or alveolar bone results inenhanced tooth and/or alveolar bone growth on areas where deteriorationhas occurred, or to prevent such deterioration.

A further aspect of the invention is a small adhesive patch forapplication on gum tissue of an individual, the patch comprising atherapeutically effective amount of a compound of the invention. Thecompound is slowly released from the patch into the gum, so that thereleased compound penetrates into the root of the teeth as well as thealveolar and/or jaw bones to prevent loss of such bones and/or toregenerate such bones.

An object of the invention is to provide a method of treating orpreventing skeletal bone or dental bone loss by theadministration/application of any formulation/composition of theinvention.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the subject invention, as more fully described below.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between bone mass and age inhumans.

FIG. 2 is a schematic drawing of a matrix extracellularphosphoglycoprotein wherein the area designated as “A” includessequences which match peptides of the present invention and the areadesignated as “B” is a highly homologous motif to a group of bone-toothmatrix phosphoglycoproteins such as osteopontin (OPN), dentinsialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bonesialoprotein II (IBSP).

FIGS. 3A, 3B, 3C, and 3D are actual photographs of bone cross-sections(from a seven day mouse calvaria organ culture study) showing theeffects of a control (FIG. 3A), fibroblast growth factor-1 (FGF-1) (FIG.3B), and two peptides of the invention designated D-00004 and D-00006(FIGS. 3C and 3D, respectively).

FIG. 4 is a graph comparing the effects of different compounds oncalvaria.

FIG. 5 is a graph showing the in vivo effects of D-00006.

FIG. 6 is a graph showing the effect of D-00006 on urinary phosphateleakage.

DETAILED DESCRIPTION OF THE INVENTION

Before the peptides, analogs, formulations, and methodology of thepresent invention are described, it is to be understood that thisinvention is not limited to any particular embodiment described, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is with the purpose of describing particular embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of such peptides and reference to “themethod” includes reference to one or more methods and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “peptide” and “peptidic compound” are used interchangeablyherein to refer to a polymeric form of amino acids of from about 10 toabout 50 amino acids, which can comprise coded and non-coded aminoacids, chemically or biochemically modified or derivatized amino acids,L- or D-amino acids, peptides having modified peptide backbones, andpeptides comprising amino acid analogs. The peptidic compounds may bepolymers of: (a) naturally occurring amino acid residues; (b)non-naturally occurring amino acid residues, e.g. N-substitutedglycines, amino acid substitutes, etc.; or (c) both naturally occurringand non-naturally occurring amino acid residues/substitutes. In otherwords, the subject peptidic compounds may be peptides or peptoids.Peptoid compounds and methods for their preparation are described in WO91/19735, the disclosure of which is herein incorporated by reference.

The terms “treat”, “treating”, “treatment” and the like are usedinterchangeably herein and mean obtaining a desired pharmacologicaland/or physiological effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of partially or completely curing a diseaseand/or adverse effect attributed the disease such as enhancing theeffect of vitamin D. “Treating” as used herein covers treating a diseasein a vertebrate and particularly a mammal and most particularly a human,and includes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e. arresting its development; or

(c) relieving the disease, i.e. causing regression of the disease.

The invention is particularly directed towards peptides which make itpossible to treat patient's which have experienced bone loss or whichwould be expected to experience bone loss and thus is particularlydirected towards preventing, inhibiting, or relieving the effects ofbone loss. A subject is “treated” provided the subject experiences atherapeutically detectable and beneficial effect which may be measuredbased on a variety of different criteria including increased bonegrowth, increased bone strength or other characteristics generallyunderstood by those skilled in the art to be desirable with respect tothe treatment of diseases related to bone.

The term “antibody” is meant an immunoglobulin protein capable ofbinding an antigen. The term “antibody” as used herein is intended toinclude antibody fragments (e.g. F(ab′)₂, Fab′, and Fab) capable ofbinding an antigen or antigenic fragment of interest.

The term “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific peptide—specifically a peptide ofthe invention. Antibody binding to its specific target epitope isstronger than the binding of the antibody to other epitopes on thepeptide or to other epitopes on other peptides. Antibodies which bindspecifically to a peptide of interest may be capable of binding to otherpeptides at a weak, yet detectable level (e.g. 10% or less of thebinding shown to the peptide of interest). Such weak binding orbackground binding, is readily discernable from the specific antibodybinding to the peptide of interest, e.g. by the use of appropriatecontrols.

The term “skeletal loss” refers to any situation in which skeletal mass,substance or matrix or any component of the skeleton, such as calciumand phosphate, is decreased or the bone is weakened such as in terms ofits ability to resist being broken.

The term “skeleton” includes both bone and teeth. In the same manner,the term “skeletal” means both bone and teeth.

The term “osteoporosis” is intended to refer to any condition involvingbone loss, i.e. involving a reduction in the amount of bone mass orsubstance resulting from any cause. The term particularly results in abone loss resulting from demineralization of the bone, post menopausalor peri-menopausal estrogen decrease or nerve damage.

The terms “subject,” “individual,” “patient,” and “host” are usedinterchangeably herein and refer to any vertebrate, particularly anymammal and most particularly including human subjects, farm animals, andmammalian pets.

Peptidic Compounds

A peptidic compound of the invention is a peptide comprising from 10 to50 amino acids. The amino acids are preferably one of the twentynaturally occurring L-amino acids. However, D-amino acids may be presentas may amino acid analogs. A peptide of the invention will comprise oneor more of the following amino acid sequence motifs: an integrin bindingmotif such as RGD sequence; a glycosaminoglycan binding motif; and acalcium binding motif. Individual amino acids may be present in thepeptides in either the L or the D isoform, but preferably in the L form.A peptide of the invention can be amidated or non-amidated on itsC-terminus, or carboxylated or non-carboxylated on its N-terminus. Thepeptide of the invention may or may not contain a glycosaminoglycanbinding motif such as SGDG (SEQ ID NO:41) sequence in L- or D-isomerform. A compound of the invention is still further characterized bybiological activity i.e. it enhances skeletal growth as well as thegrowth or recruiting of osteoblast or odontoblast cells on surface ofthe new skeletal growth.

A peptidic compound of the invention exhibit one or more of thefollowing properties when administered in an effective amount to anindividual: (1) reduce bone loss; (2) increase bone mass; (3) increasebone strength; (4) reduce renal excretion of phosphate; and (5) reduceloss of phosphate from an individual.

Specific examples of peptides of the invention comprise seven toforty-seven amino acids on either side of the RGD sequence of thenaturally occurring sequence of matrix extracellularphosphoglycoprotein. Thus, examples of peptides of the inventioncomprising sequences taken from the following sequence and including theRGD sequence shown in bold:

(SEQ ID NO:1) DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQERGDNDISPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFAGPSEAESTHL

Specific examples of peptides of the invention which comprise the RGDsequence as the terminal sequence include the following:

(SEQ ID NO:2) AQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQERGD (SEQ IDNO:3) RGDAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:4)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDRGD (SEQ ID NO:5)RGDSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:6)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFEGSGRGD (SEQ ID NO:7)RGDTHRIQHNIDYLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:8)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSDFERGD (SEQ ID NO:9)RGDLKHLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:10)DSQAQKSPVKSKSTHRIQHNIDYLKHLSKVKKIPSRGD (SEQ ID NO:11)RGDLSKVKKIPSDFEGSGYTDLQE (SEQ ID NO:12) DSQAQKSPVKSKSTHRIQHNIDYLKHLSKRGD(SEQ ID NO:13) RGDVKKIPSDFEGSGYTDLQE (SEQ ID NO:14)DSQAQKSPVKSKSTHRIQHNIDYLKRGD (SEQ ID NO:15) RGDIPSDFEGSGYTDLQE (SEQ IDNO:16) DSQAQKSPVKSKSTHRIQHNIDRGD (SEQ ID NO:17) RGDDFEGSGYTDLQE (SEQ IDNO:18) DSQAQKSPVKSKSTHRRGD (SEQ ID NO:19) RGDGSGYTDLQE (SEQ ID NO:20)DSQAQKSPVKRGD (SEQ ID NO:21) RGDGYTDLQE (SEQ ID NO:22) DSQAQKSRGD (SEQID NO:23) RGDNDISPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA

Specific examples of the peptides of the invention which comprise theRGD internally include the following:

(SEQ ID NO:24) NDI RGDSPFSGDGQPFKDIPGKGEATGPDLEGKDIQTGFA (SEQ ID NO:25)NDISPF RGDSGDGQPFKDIPGKGEATGPDLEGKDI (SEQ ID NO:26) NDISPFSGDRGDGQPFKDIPGKGEATGPDL (SEQ ID NO:27)FSGDGQPFKDIPGKGEATGPDLEGKDIQTGFAGPSEAES RGDTHL (SEQ ID NO:28)IPGKGEATGPDLEGKDIQTGFAGPSE RGDAESTHL (SEQ ID NO:29) EATGPDLEGKDIQTGFAGRGDPSEAESTHL (SEQ ID NO:30) NDISPFSGDGQPFKD RGDIPGKGEATGPDLEGK (SEQ IDNO:31) GKGEATGPDLEGKDI RGDQTGFAGPSEAESTHL (SEQ ID NO:32)FSGDGQPFKDIPGKGEATG RGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:33)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:34) PFKDIPGKGEATGRGDPDLEGKDIQ (SEQ ID NO:35) DIPGKGEATG RGDPDLEGKDIQTGFAGP (SEQ ID NO:36)DGQPFKDIPGKGEATG RGDPDLEGKDIQTGF (SEQ ID NO:37) GKGEATGRGDPDLEGKDIQTGFAGPSEA (SEQ ID NO:38) EATG RGDPDLEGKDIQTGF (SEQ ID NO:39)EATG RGDPDLEGK (SEQ ID NO:40) EATG RGDPDL

In some embodiments, a peptide of the invention comprises aglycosaminoglycan-binding motif. A glycosaminoglycan binding motif hasthe consensus sequence SGXG (SEQ ID NO:50), wherein X is any amino acid.In some embodiments, a glycosaminoglycan binding motif has the sequenceSGDG (SEQ ID NO:41).

In other embodiments, a peptide of the invention comprises a calciumbinding motif. In some embodiments, a calcium binding motif has thesequence DNDISPFSGDGQ (SEQ ID NO:42). Also included in the term “calciumbinding motif” are amino acid sequences that differ from SEQ ID NO:42 byone, two, three, four, five, six, seven, or eight amino acids. Ofparticular interest in many embodiments are motifs that conserve aminoacids 1, 3, 5, 7, 9, and 12 of SEQ ID NO:42. Thus, in some embodiments,a peptide of the invention comprises, as a calcium-binding motif, thesequence DXDXSXFXGXXQ (SEQ ID NO:43), wherein X is any amino acid oramino acid analog.

In other embodiments, a calcium binding motif has the sequence

-   DX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂, wherein:-   X₁ is any amino acid;-   X₂ is D, N, or S;-   X₃ is I, L, V, F, Y, or W;-   X₄ is D, E, N, S, T, or G;-   X₅ is D, N, Q, G, H, R, or K;-   X₆ is G or P;-   X₇ is L, I, V, M, C;-   X₈ is D, E, N, Q, S, T, A, G, or C;-   each of X₉ and X₁₀ is independently any amino acid;-   X₁₁ is D or E; and-   X₁₂ is L, I, V, M, F, Y, or W.

In other embodiments, a calcium binding motif has the sequenceX₁X₂X₃X₄C(X₅)_(n)C(X₆)_(m)CX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄C, wherein

-   each of X₁ , X₃, and X₄ is independently D, E, Q, or N;

each of X₂, X₅, X₆, X₇, X₉, X₁₀, X₁₁, X₁₂, and X₁₄ is independently anyamino acid;

-   n is 3-14;-   m is 3-7;-   X₈ is D or N; and-   X₁₃ is F or Y.

In other embodiments, a calcium binding motif has the sequenceX₁X₂X₃X₄X₅DX₆X₇X₈X₉X₁₀X₁₂X13X14X15X16X17X₁₈X₁₉X₂₀X₂₁, wherein each of X₁and X₂ is independently L, I, V, M, F, Y, or W;

-   each of X₃, X₄, X₆, X₇, X₈, X₁₀, X₁₁, X₁₂, X₁₅, X₁₈, and X₁₉ is    independently any amino acid;-   X₅ is L or K;-   X₉ is D or N;-   X₁₃ is D, N, S, or G;-   X₁₄ is F or Y;-   X₁₆ is E or S;-   X₁₇ is F, Y, V, or C;-   X₂₀ is L, I, V, M, F, or S;-   and X₂₁ is L, I, V, M, or F.

In other embodiments, a calcium binding motif has the sequenceDX₁X₂X₃X₄X₅X₆GX₇DX₈X₉X₁₀GGX₁₁X₁₂X₁₃D, wherein each of X₁, X₃, X₄, X₅,X₆, X₇, X₈, X₁₀, X₁₁, X₁₂, and X₁₃ is independently any amino acid; and

each of X₂ and X₉ is independently L or I.

Calcium binding motifs are known in the art and have been describedamply. See, for example, Springer et al. (2000) Cell 102:275-277;Kawasaki and Kretsinger (1995) Protein Prof 2:305-490; Moncrief et al.(1990 J. Mol. Evol. 30-522-562; Chauvaux et al. (1990) Biochem. J.265:261-265; Bairoch and Cox (1990) FEBS Lett. 269:454-456; Davis (1990)New Biol. 2:410-419; Schaefer et al. (1995) Genomics 25:638-643; andEconomou et al. (1990) EMBO J. 9 :349-354. Any known calcium bindingmotif can be included in a peptidic compound of the invention.

A peptide of the invention may comprise one or more of an integrinbinding motif, a glycosaminoglycan binding motif, and a calcium bindingmotif. The motifs may be present in the peptide in any order relative toone another. The motifs may be separated from one another by one, two,three, four, five, six, seven, eight, nine, or ten amino acids, or more.Furthermore, a motif may overlap with one or more other motifs. As onenon-limiting example, a peptide having the sequenceTDLQERGDNDISPFSGDGQPFKD (SEQ ID NO:49) comprises all three motifs, whichoverlap with one another.

All or any of the amino acids in the above sequences may be in the D- orL-conformation and may be substituted with equivalent analogs. Thepreferred embodiments comprise naturally occurring amino acids in theL-conformation.

All or any of the above sequences may be amidated, non-amidated, orotherwise modifed on their C-terminus, or carboxylated,non-carboxylated, or otherwise modified on their N-terminus.

In addition, multimers of any of the foregoing peptides are provided.Multimers include dimers, trimers, tetramers, pentamers, hexamers, etc.Thus, a peptide of the invention having a length of from about 10 toabout 50 amino acids can be multimerized, optionally with an interveninglinker, such that a subject peptide occurs in tandem arrays of two,three, four, five, six, or more copies. Furthermore, two or moredifferent peptides of the invention can be multimerized with oneanother, forming “heteromultimers.” Thus, e.g., a multimer may comprisea first and a second peptide, linked together by peptide bonds,optionally with a linker molecule such as one to ten glycine residues.

Peptidic compounds of the invention can be obtained using any knownmethod, including, e.g., solid phase peptide synthesis techniques, wheresuch techniques are known to those of skill in the art. Methods forsynthesizing peptides are well known in the art and have been amplydescribed in numerous publications, including, e.g., “The Practice ofPeptide Synthesis” M. Bodanszky and A. Bodanszky, eds. (1994)Springer-Verlag; and Jones, The Chemical Synthesis of Peptides(Clarendon Press, Oxford)(1994). Generally, in such methods a peptide isproduced through the sequential additional of activated monomeric unitsto a solid phase bound growing peptide chain. Also of interest is theuse of submonomers in solid phase synthesis, as described in WO94/06451, the disclosure of which is herein incorporated by reference.

Instead of solid phase synthesis, the subject peptidic compounds of thesubject invention may be prepared through expression of an expressionsystem comprising a polynucleotide encoding the peptidic compound. Anyconvenient methodology may be employed, where methodologies that may beemployed typically include preparation of a nucleic acid moleculecomprising a nucleotide sequence encoding the subject peptide,introduction of the encoding region into a vector for expression,transformation of a host cell with the vector, and expression andrecovery of the product. Protocols for accomplishing each of the abovesteps are well known in art. See Sambrook, Fritsch & Maniatis, MolecularCloning, A Laboratory Manual (Cold Spring Harbor Press, Inc.)(1989).

Matrix extracellular phosphoglycoprotein was cloned and characterizedfrom a human tumor that caused osteomalacia in the patients. Thisextremely rare type of tumor called Oncogenic HypophosphatemicOsteomalacia (OHO) tumor has been known to cause renal phosphate leak,hypophosphatemia (low serum phosphate levels), low serum calcitriol(1,25-vitamin D3), and abnormalities in skeletal mineralization(Osteomalacia). In the patients of OHO tumor, resection of the tumorsresults in remission of all of the above symptoms and it has beenproposed that a circulating phosphaturic factor secreted from OHO tumorplays a role in osteomalacia. Matrix extracellular phosphoglycoproteinwas proposed as a candidate of this phosphaturic factor (Rowe et. al.,Genomics (2000) 67:56-68).

Phosphate plays a central role in many of the basic processes essentialto the cell and the mineralization of skeleton. In particular, skeletalmineralization is dependent on the regulation of phosphate and calciumin the body and any disturbances in phosphate-calcium homeostasis canhave severe repercussions on the integrity of bone. In the kidney,phosphate is lost passively into the glomerular filtrate and is activelyreabsorbed via a sodium (Na+) dependent phosphate cotransporter. In theintestine, phosphate is absorbed from foods. A sodium (Na+) dependentphosphate cotransporter was found to be expressed in the intestine andrecently cloned (Hilfiker, PNAS 95(24) (1998), 14564-14569). The liver,skin and kidney are involved in the conversion of vitamin D3 to itsactive metabolite, calcitriol, which plays an active role in themaintenance of phosphate balance and skeletal mineralization.

Vitamin D deficiency causes rickets in children and osteomalacia inadults. Both conditions are characterized by failure of calcification ofosteoid, which is the matrix of skeleton.

Thus, all of the humoral functions by matrix extracellularphosphoglycoprotein, namely, renal phosphate leak, hypophosphatemia (lowserum phosphate levels), low serum calcitriol (1,25-vitamin D3), areharmful to healthy skeletal formation.

Matrix extracellular phosphoglycoprotein is a large polypeptide with 525amino acid with short N-terminus signal sequence. Therefore, it ishighly probable that this molecule is secreted from its producing cellsinto the body fluid and circulation. Out of its 525 amino acid sequence,a 23 amino acid motif on the C-terminus showed high similarities to agroup of bone-tooth mineral matrix phosphoglycoproteins such asosteopontin (OPN), dentin sialophosphoprotein (DSPP), dentin matrixprotein 1 (DMP1), and bone sialoprotein II (IBSP). It has been proposedthat these bone-tooth mineral matrix phosphoproteins may play importantroles in skeletal mineralization.

Notwithstanding the above observations about matrix extracellularphosphoglycoprotein, smaller peptide sequence containing integrinbinding motif that is located within the amino acid sequence and farfrom its C-terminus sequence with a high degree of similarity to otherbone-tooth mineral matrix phosphoglycoproteins demonstrated a verypotent skeletal formation activity and increased the number ofosteoblasts on such skeletal formation surface. The potency of suchactivities was equivalent to fibroblast growth factor (FGF). It wassurprising in that small motifs located within a larger protein whichhas destructive functions on the skeleton demonstrated potent boneformation activity, and that such motifs were located far from thesequence which showed homology to other known bone-tooth matrixproteins.

Another surprising fact was that potent skeletal formation motifs of theinvention contained an integrin binding motif, in particular, RGDsequence. It has been reported that a synthetic peptide containing theRGD sequence inhibited bone formation and resorption in a mineralizingorgan culture system of fetal rat skeleton (Gronowicz et. al. Journal ofBone and Mineral Research 9(2):193-201 (1994)), that is a very similarexperimental method used to test the subject of the present invention.

Further, the skeletal formation activity provided by the small peptidesof the invention was as potent as that of an intact growth factor suchas FGF.

Therapeutic Methods

The invention provides methods for reducing skeletal bone loss, methodsfor reducing renal phosphate leakage, methods for increasing bone mass,methods for increasing bone strength, and methods for reducing Piexcretion, comprising administering a peptidic compound of theinvention. Typically, a peptidic compound of the invention is formulatedwith a pharmaceutically acceptable excipient for delivery to anindividual in need thereof.

As used herein, an “effective amount” of a peptidic compound of theinvention is an amount that reduces bone loss, and/or increases bonestrength, and/or increases bone mass, and/or reduces phosphate loss,and/or reduces renal phosphate excretion by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, or at least about 60%, or more, whencompared to a suitable control. Suitable controls are, in the case ofexperimental animals, an animal not treated with the peptide, e.g.,treated with vehicle, or treated with an irrelevant peptide; and in thecase of human subjects, a human subject treated with a placebo, or ahuman subject before treatment with a peptide of the invention.

In some embodiments, an effective amount of a peptidic compound of theinvention is an amount that reduces renal phosphate excretion, andtherefore reduces phosphate loss from an individual, by at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, or at least about 60%, ormore, when compared to a suitable control.

Whether a given peptide reduces bone loss, and/or increases bonestrength, and/or increases bone mass, and/or reduces phosphate loss,and/or reduces renal phosphate excretion in an individual can bedetermined using any known assay to measure any known parameterassociated with any one or more of reduced bone loss, increased bonestrength, increased bone mass, reduces phosphate loss, and reduced renalphosphate excretion, including, but not limited to, serum and urinaryphosphorus levels (e.g., using a colorimetric assay); serum and urinarycalcium levels (e.g., using a colorimetric assay); serum and urinarycreatinine levels; bone turnover marker levels (e.g., deoxypyrodinolineand osteocalcin); bone density (e.g., by in vivo bone densitometry);bone mechanical testing (e.g., lumbar vertebrae compression test;femoral shaft three point bending test; and the like); and the like.Such methods are standard in the art.

Individuals suitable for treatment with the methods of the invention areindividuals having to believed to be at risk for, bone loss, or adisorder caused by bone loss, or a disorder whose sequelae include boneloss, including, but not limited to, dental caries, osteoporosis, Paget's disease, renal phosphate leakage, renal osteodystrophy, osteomalacia,osteodystrophy resulting from other causes, osteolysis mediated bycancer, fractures, and hyperparathyroidism. Such individuals includeolder individuals, post-menapausal women, kidney transplant recipients,and individuals having, or being at risk for, any of the aforementioneddisorders.

Routes of Administration

Peptides of the invention are administered to an individual using anyavailable method and route suitable for drug delivery, including in vivoand ex vivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, topical application, intravenous, rectal, nasal, oral andother parenteral routes of administration. Routes of administration maybe combined, if desired, or adjusted depending upon the immunomodulatorynucleic acid molecule and/or the desired effect on the immune response.Peptides of the invention can be administered in a single dose or inmultiple doses.

Peptides of the invention can be administered to a subject using anyavailable conventional methods and routes suitable for delivery ofconventional drugs, including systemic or localized routes. In general,routes of administration contemplated by the invention include, but arenot necessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of peptides of theinvention. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

Peptides of the invention can also be delivered to the subject byenteral administration. Enteral routes of administration include, butare not necessarily limited to, oral and rectal (e.g., using asuppository) delivery.

Methods of administration of a peptide of the invention through the skinor mucosa include, but are not necessarily limited to, topicalapplication of a suitable pharmaceutical preparation, transdermaltransmission, injection and epidermal administration. Also contemplatedfor delivery of a peptide of the invention is a patch containing thereina peptide of the invention. A patch can be applied to the skin, or toother tissue, e.g., gum tissue. Any known patch delivery system that issuitable for oral delivery system can be used. See, e.g., U.S. Pat. No.6,146,655.

Peptides of the invention can also be delivered to an individual byadministering to the individual a nucleic acid molecule comprising anucleotide sequence that encodes a peptide of the invention. The terms“polynucleotide” and “nucleic acid molecule” are used interchangeablyherein to refer to polymeric forms of nucleotides of any length. Thepolynucleotides may contain deoxyribonucleotides, ribonucleotides,and/or their analogs. For expression, an expression cassette may beemployed. The expression vector will provide a transcriptional andtranslational initiation region, which may be inducible or constitutive,where the coding region is operably linked under the transcriptionalcontrol of the transcriptional initiation region, and a transcriptionaland translational termination region. These control regions may benative to a gene encoding the subject peptides, or may be derived fromexogenous sources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor the production of fusion proteins, where the exogenous fusionpeptide provides additional functionality, i.e. increased proteinsynthesis, stability, reactivity with defined antisera, an enzymemarker, e.g. β-galactosidase, etc.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Vectors include, but are not limited to, plasmids;cosmids; viral vectors; artificial chromosomes (YAC's, BAC's, etc.);mini-chromosomes; and the like. Vectors are amply described in numerouspublications well known to those in the art, including, e.g., ShortProtocols in Molecular Biology, (1999) F. Ausubel, et al., eds., Wiley &Sons.

Expression vectors may be used to introduce a nucleic acid moleculeencoding a subject peptide into a cell of an individual. Such vectorsgenerally have convenient restriction sites located near the promotersequence to provide for the insertion of nucleic acid sequences.Transcription cassettes may be prepared comprising a transcriptioninitiation region, the target gene or fragment thereof, and atranscriptional termination region. The transcription cassettes may beintroduced into a variety of vectors, e.g. plasmid; retrovirus, e.g.lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

An expression vector comprising a nucleotide sequence encoding a peptideof the invention may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The expression vector may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the expression vector, then bombarded into skin cells.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1Φg, to about 1,000 Φg, to about 10,000 Φg, to about 25,000 Φg or about50,000 Φg of a peptide of the invention. Peptides of the invention canbe administered in a single dosage or several smaller dosages over time.Alternatively, a target dosage of a peptide can be considered to beabout 0.1-1000 μM, about 1-500 ΦM, or about 5-250 μM in a sample of hostblood drawn within the first 24-48 hours after administration of thepeptide.

The effect on bone loss, bone strength, phosphate excretion, or otherparameter may be dose-dependent. Therefore, to increase potency by amagnitude of two, each single dose is doubled in concentration.Increased dosages may be needed to achieve the desired therapeutic goal.The invention thus contemplates administration of multiple doses toprovide and maintain an effect on bone loss, bone strength, phosphateexcretion, or other parameter. When multiple doses are administered,subsequent doses are administered within about 16 weeks, about 12 weeks,about 8 weeks, about 6 weeks, about 4 weeks, about 2 weeks, about 1week, about 5 days, about 72 hours, about 48 hours, about 24 hours,about 12 hours, about 8 hours, about 4 hours, or about 2 hours or lessof the previous dose.

In view of the teaching provided by this disclosure, those of ordinaryskill in the clinical arts will be familiar with, or can readilyascertain, suitable parameters for administration of peptides accordingto the invention.

Formulations

In general, peptides are prepared in a pharmaceutically acceptablecomposition for delivery to a host. Pharmaceutically acceptable carrierspreferred for use with the peptides of the invention may include sterileaqueous of non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. A composition comprising a peptide of the invention may alsobe lyophilized using means well known in the art, for subsequentreconstitution and use according to the invention. Also of interest areformulations for liposomal delivery, and formulations comprisingmicroencapsulated peptides.

In general, the pharmaceutical compositions can be prepared in variousforms, such as granules, tablets, pills, suppositories, capsules,suspensions, salves, lotions and the like. In some embodiments, wheredelivery of a peptide of the invention is to oral tissues, a peptide ofthe invention may be formulated in a toothpaste, a mouthwash, or may becoated on or embedded in a dental floss. Pharmaceutical grade organic orinorganic carriers and/or diluents suitable for oral and topical use canbe used to make up compositions comprising the therapeutically-activecompounds. Diluents known to the art include aqueous media, vegetableand animal oils and fats. Stabilizing agents, wetting and emulsifyingagents, salts for varying the osmotic pressure or buffers for securingan adequate pH value, and skin penetration enhancers can be used asauxiliary agents. Preservatives and other additives may also be presentsuch as, for example, anti-pathogenic agents (e.g., antimicrobials,antibacterials, antivirals, antifungals, etc.), antioxidants, chelatingagents, and inert gases and the like.

A peptidic compound of the invention can be administered with any otherknown agent that reduces bone loss. Thus, combination therapy iscontemplated. Other agents that can be administered with a peptide ofthe invention include, but are not limited to, estrogen, calcitonin,vitamin D, fluoride, Ipriflavon, and bisphosphonate. A peptide of theinvention can be administered simultaneously with (e.g., in admixturewith, or in separate formulations) another agent that reduces bone loss;or can be administered within about 15 minutes, about 30 minutes, about60 minutes, about 2 hours, about 5 hours, about 10 hours, about 12hours, about 24 hours, about 36 hours, about 4 days, about 7 days, ormore, of another agent that reduces bone loss. In addition, two or morepeptides of the invention can be administered simultaneously or withinabout 15 minutes, about 30 minutes, about 60 minutes, about 2 hours,about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36hours, about 4 days, about 7 days, or more of each other.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Synthesis of D-00001, etc.

Six different peptides were manually synthesized by the9-fluorenylmethoxycarbonyl (Fmoc) strategy and prepared in theC-terminal amide form. The six peptides are as follows:

D-00001: IPSDFEGSGYTDLQE (SEQ ID NO:44) D-00002: DFEGSGYTDLQERGD (SEQ IDNO:45) D-00003: YTDLQERGDNDISPF (SEQ ID NO:46) D-00004: ERGDNDISPFSGDGQ(SEQ ID NO:47) D-00005: NDISPFSGDGQPFKD (SEQ ID NO:48) D-00006:TDLQERGDNDISPFSGDGQPFKD (SEQ ID NO:49) (C-terminus amidated)

Amino acid derivatives and resins were purchased from Bachem, Inc.,Torrance, Calif., and Novabiochem, La Jolla, Calif. The respective aminoacids were condensed manually in a stepwise manner using 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin. N-methyl pyrrolidonewas used during the synthesis as a solvent. For condensation,diisopropylcarbodiimide/N-hydroxybenzotriazole was employed, and fordeprotection of N^(α) -Fmoc groups, 20% piperidine in N-methylpyrrolidone was employed. The following side chain protecting groupswere used: Asn and Gln, trityl; Asp, Glu, Ser, and Thr, t-butyl; Arg,2,2,5,7,8-pentamethylchroman-6-sulfonyl; and Lys, t-butoxycarbonyl.Resulting protected peptide resins were deprotected and cleaved from theresin using a trifluoroaceticacid-thioanisole-m-cresol-ethanedithiol-H₂O (80:5:5:5:5, v/v) at 20° C.for 2 h. Resulting crude peptides were precipitated and washed withethyl ether then purified by reverse-phase high performance liquidchromatography (using Vydac 5C18 column and a gradient ofwater/acetonitrile containing 0.1% trifluoroacetic acid). All peptideswere obtained with 5-20% yield (from the starting resin). Purity of thepeptides was confirmed by analytical high performance liquidchromatography. Identity of the peptides was confirmed by a Sciex APIIIIE triple quadrupole ion spray mass spectrometer.

Example 2 Fetal Mouse Calvarial Assay Reagents

FGF-1 was purchased from Peprotech Inc. (Rocky Hill, N.J.). RGD-1, 2, 3,4, 5 and 6 (referred to here as D-00001, D-00002, D-00003, D-00004,D-00005 and D-00006) were provided by Dr. Nomizu (Hokkaido University,Japan).

Mice

Pregnant ICR mice were purchased from SLC Japan Co. Ltd. (Shizuoka,Japan).

Mouse Calvarial Organ Culture

Mouse calvarial organ culture was performed as described in Mundy G etal. Science 286: 1946-1949, 1999 and Traianedes K et al. Endocrinology139: 3178-3184, 1998. The calvaria from 4-days-old mice were excised andcut in half along the sagittal suture. Each half of the calvaria wasplaced on a stainless steel grid in a 12-well tissue culture dish (AsahiGlass Techno Corp., Funabashi, Japan). Each well contained 1.5 ml of BGjmedium (Sigma, St. Louis, Mo.) supplemented with 0.1% bovine serumalbumin (Sigma) and each compound. FGF-1 was used as a positive controlas described by Mundy et al. The medium was changed at day 1 and 4, andthe assay was terminated at day 7.

Histomorphometrical Analysis

Calvaria was fixed with 10% neutral-buffered formalin, decalcified with4.13% EDTA and embedded in paraffin. 4 mm-thickness sections were madeand stained with hematoxylin and eosin. New bone area was measured usingImage-Pro Plus (Media Cybernetics, Silver Spring, Md.).

The six peptides of Example 1 were tested for their ability to enhancebone growth with the tests being carried out as described above inExample 2. The peptides which did not include the RGD sequence did notshow positive results. The other four peptides showed positive resultswith the best results being obtained with the sequences

D-00004: ERGDNDISPFSGDGQ, (SEQ ID NO:47) and D-00006:TDLQERGDNDISPFSGDGQPFKD. (SEQ ID NO:49)

The best results are in FIG. 3 (specifically FIG. 3C and 3D). Data fromthese results are graphically shown in FIGS. 4.

Example 3 In Vivo Bone Formation Study Reagents

FGF-1 was purchased from Peprotech Inc. (Rocky Hill, N.J.). RGD-6(referred to here as D-00006) was synthesized by CS Bio (San Carlos,Calif.) under the instruction of the inventors.

D-00006: TDLQERGDNDISPFSGDGQPFKD. (SEQ ID NO:49)

Mice

Four week old mice were purchased from SLC Japan Co. Ltd. (Shizuoka,Japan) and randomized to three groups (n=5).

Mouse Calvaria Growth Assay

D-00006 (20 μg/kg/day), FGF-1 (12.5 μg/kg/day), or vehicle (saline) wassubcutaneously injected into the soft tissues adjacent to the calvariaeof the test animals. The daily amount of the samples were divided bytwo, respectively, and injected twice a day for five days. After 15 daysof the last administration, the calvariae were excised and cut in halfalong the sagittal suture, and provided to the Histomorphometricalanalysis.

Histomorphometrical Analysis

Calvaria was fixed with 10% neutral-buffered formalin, decalcified with4.13% EDTA and embedded in paraffin. 4mm-thickness sections were madeand stained with hematoxylin and eosin. New bone area was measured usingImage-Pro Plus (Media Cybernetics, Silver Spring, Md.).

Results

The calvaria sections from the animals treated with FGF-1 showed asignificant expansion of bone area as compared to the vehicle treatedgroup. Those from the animals treated with D-00006 also demonstrated asignificant expansion of bone area as compared to the vehicle treatedgroup and the efficacy was equivalent to that of FGF-1. Data from theseresults are graphically shown in FIG. 5.

Example 4 Effect of D-00006 on Renal Phosphate Excretion ExperimentalDesign and Treatments

Forty 3-month old virgin female Sprague-Dawley rats (Harlan SpragueDawley, Inc.) were acclimated for one week prior to beginning theexperiments. Following the acclimatization period, the animal wererandomized by initial body weight into treatment groups outlined inTable 1. Ovx: ovariectomized; LD: low dose; HD: high dose; Est:estradiol; D-00006 (TDLQERGDNDISPFSGDGQPFKD; SEQ ID NO:49).

TABLE 1 Group # of No. Description Treatment Dose-Level Animals 1 ShamVehicle — 8 2 Ovx Vehicle — 8 3 Ovx + LD D-00006  20 μg/kg/day 8 4 Ovx +HD D-00006 200 μg/kg/day 8 5 Ovx + Est 17 β-estradiol  10 μg/kg/day 8pellet implant and vehicle

One day prior to the initiation of treatments, the animals wereanesthetized with a ketamin/xylazine anesthetic mixture, and animals ingroups 2-5 were ovariectomized.

Urine was collected in metabolic cages on Day 41. Blood and urinesamples were collected for chemistry and bone turnover markers assays atthe end of the 41-day treatment period. Prior to urine and bloodcollections, the animals were placed in metabolic cages and deprived offood for an overnight fast period of 18 hours. Urine samples werecollected and the volumes recorded. The urine samples were thencentrifuges at approximately 3000×g for 10 minutes in a refrigeratedcentrifuge. The samples were filtered to remove contaminating sediments.

Serum and Urine Chemistry

Total serum calcium and creatinine levels were the same in all treatmentgroups, except that the Ovx animals treated with estradiol showed aslight increase in serum calcium. There was a dose-dependent increase inserum phosphorus in the D-0006 treated groups. Total urine volumes,collected over an 18-hour period, were the same in all groups. FIG. 6shows the derived urine parameters.

The total amount of phosphorus in the 18-hour urine collections showed asignificant decrease in D-00006 treated groups. As a result, D-00006treated groups demonstrated lower phosphate clearance and a higherpercentage of tubular reabsorption of phosphorus. D-00006 was clearlyshown to be an agent that conserves phosphorus in the circulation.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-13. (canceled)
 14. A method of reducing bone loss, comprising:administering to an individual a therapeutically effective amount of aformulation comprising: a carrier; and a peptide compound comprising atleast 10 and not more than 50 naturally occurring amino acids thepeptide compound comprising an integrin binding motif, aglycosaminoglycan binding motif, and a calcium binding motif.
 15. Themethod of claim 14, wherein the integrin binding motif is an RGDsequence.
 16. The method of claim 14, wherein the glycosaminoglycanmotif has the sequence SGDG.
 17. The method of claim 14, wherein thecalcium binding motif has the sequence DXDXSXFXGXXQ, wherein X is anyamino acid.
 18. The method of claim 17, wherein the calcium bindingmotif has the sequence DNDISPFSGDGQ.
 19. The method of claim 14, whereinthe carrier is a saline solution and the formulation is injectable. 20.The method of claim 14, wherein the peptide comprises an amino acidsequence contiguous with a RGD sequence of naturally occurring matrixextracellular phosphoglycoprotein.
 21. The method of claim 14, whereinthe peptide compound is a multimer.
 22. A patch for transdermal deliveryof a peptide, a patch comprising: a carrier; and a peptide compoundcomprising at least 10 and not more than 50 naturally occurring aminoacids the peptide compound comprising an integrin binding motif, aglycosaminoglycan binding motif, and a calcium binding motif.
 23. Amethod of reducing renal phosphate excretion in an individual,comprising: administering to an individual a therapeutically effectiveamount of a formulation comprising: a carrier; and a peptide compoundcomprising at least 10 and not more than 50 naturally occurring aminoacids the peptide compound comprising an integrin binding motif, aglycosaminoglycan binding motif, and a calcium binding motif.
 24. Themethod of claim 23, wherein the integrin binding motif is an RGDsequence.
 25. The method of claim 23, wherein the glycosaminoglycanmotif has the sequence SGDG.
 26. ( The method of claim 23, wherein thecalcium binding motif has the sequence DXDXSXFXGXXQ, wherein X is anyamino acid.
 27. The method of claim 26, wherein the calcium bindingmotif has the sequence DNDISPFSGDGQ.
 28. The method of claim 23, whereinthe carrier is a saline solution and the formulation is injectable.